Chemically based transmissible ER stress protocols are unsuitable to study cell-to-cell UPR transmission

Renal epithelial cells regulate the destructive activity of macrophages and participate in the progression of kidney diseases. Critically, the Unfolded Protein Response (UPR), which is activated in renal epithelial cells in the course of kidney injury, is required for the optimal differentiation and activation of macrophages. Given that macrophages are key regulators of renal inflammation and fibrosis, we suppose that the identification of mediators that are released by renal epithelial cells under Endoplasmic Reticulum (ER) stress and transmitted to macrophages is a critical issue to address. Signals leading to a paracrine transmission of ER stress (TERS) from a donor cell to a recipient cells could be of paramount importance to understand how ER-stressed cells shape the immune microenvironment. Critically, the vast majority of studies that have examined TERS used thaspigargin as an inducer of ER stress in donor cells in cellular models. By using multiple sources of ER stress, we evaluated if human renal epithelial cells undergoing ER stress can transmit the UPR to human monocyte-derived macrophages and if such TERS can modulate the inflammatory profiles of these cells. Our results indicate that carry-over of thapsigargin is a confounding factor in chemically based TERS protocols classically used to induce ER Stress in donor cells. Hence, such protocols are not suitable to study the TERS phenomenon and to identify its mediators. In addition, the absence of TERS transmission in more physiological models of ER stress indicates that cell-to-cell UPR transmission is not a universal feature in cultured cells.

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Super-resolution microscopy reveals that Na+/K+-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad

Cell Death and Disease ◽

10.1038/s41419-021-04025-8 ◽

2021 ◽

Vol 12 (8) ◽

Author(s):

Kristoffer Bernhem ◽

Jacopo M. Fontana ◽

Daniel Svensson ◽

Liang Zhang ◽

Linnéa M. Nilsson ◽

...

Keyword(s):

Epithelial Cells ◽

Chronic Diseases ◽

Kidney Diseases ◽

Super Resolution ◽

Diabetic Patients ◽

Diabetic Kidney ◽

Renal Epithelial Cells ◽

Apoptotic Process ◽

Induced Apoptosis ◽

Super Resolution Microscopy

AbstractActivation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na+,K+-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20 mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10 nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca2+/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.

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Refrigeration of donor cells in preparation for bovine somatic nuclear transfer

Reproduction ◽

10.1530/rep.0.1220801 ◽

2001 ◽

pp. 801-808 ◽

Cited By ~ 5

Author(s):

JL Liu ◽

MK Wang ◽

QY Sun ◽

XR Zhang ◽

LK Jiang ◽

...

Keyword(s):

Nuclear Transfer ◽

Cultured Cells ◽

Donor Cell ◽

Serum Starvation ◽

Fresh Tissue ◽

Reconstructed Embryos ◽

Donor Cells ◽

Somatic Nuclear Transfer

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.

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ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2012.06.003 ◽

2012 ◽

Vol 1823 (10) ◽

pp. 1864-1876 ◽

Cited By ~ 31

Author(s):

Wing-Kee Lee ◽

Prabir K. Chakraborty ◽

Eleni Roussa ◽

Natascha A. Wolff ◽

Frank Thévenod

Keyword(s):

Cell Death ◽

Epithelial Cells ◽

Er Stress ◽

Renal Epithelial Cells

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ERK1/2‐dependent bestrophin‐3 (Best‐3) expression prevents ER‐stress‐induced cell death of renal epithelial cells by reducing CHOP

The FASEB Journal ◽

10.1096/fasebj.26.1_supplement.1064.12 ◽

2012 ◽

Vol 26 (S1) ◽

Author(s):

Wing-Kee Lee ◽

Prabir K Chakraborty ◽

Eleni Roussa ◽

Natascha A Wolff ◽

Frank Thévenod

Keyword(s):

Cell Death ◽

Epithelial Cells ◽

Er Stress ◽

Renal Epithelial Cells

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1628: Oxalate-Induced Mitochondrial Membrane Permeability Transition and an Imbalance in the Expression/Activity of Antioxidant Enzvmes Increases Oxidative Stress and Cell Injury in Renal Epithelial Cells: Effect of Vitamine and Vitamin-C

The Journal of Urology ◽

10.1016/s0022-5347(18)31816-0 ◽

2007 ◽

Vol 177 (4S) ◽

pp. 540-540

Author(s):

Vijayalakshmi Thamilselvan ◽

Mani Menon ◽

Sivagnanam Thamilselvan

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Vitamin C ◽

Mitochondrial Membrane ◽

Cell Injury ◽

Permeability Transition ◽

Mitochondrial Membrane Permeability ◽

Renal Epithelial Cells ◽

Membrane Permeability Transition ◽

Expression Activity

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IL-17 and CD40-L act synergistically in the activation of human renal epithelial cells and can both be detected during renal allograft rejection

Immunology Letters ◽

10.1016/s0165-2478(97)88522-7 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 415

Author(s):

C van Kooten

Keyword(s):

Epithelial Cells ◽

Allograft Rejection ◽

Renal Allograft ◽

Renal Epithelial Cells ◽

Renal Allograft Rejection

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Improved micro-impedance spectroscopy to determine cell barrier properties

The EuroBiotech Journal ◽

10.2478/ebtj-2020-0017 ◽

2020 ◽

Vol 4 (3) ◽

pp. 150-155 ◽

Cited By ~ 1

Author(s):

Md. Mehadi Hasan Sohag ◽

Olivier Nicoud ◽

Racha Amine ◽

Abir Khalil-Mgharbel ◽

Jean-Pierre Alcaraz ◽

...

Keyword(s):

Impedance Spectroscopy ◽

Epithelial Cells ◽

Lipid Bilayers ◽

Cultured Cells ◽

Cell Model ◽

Measurement Techniques ◽

Cell Monolayer ◽

Microfluidic Channel ◽

Small Surface ◽

Resistance Pathway

AbstractThe goal of this study was to determine whether the Tethapod system, which was designed to determine the impedance properties of lipid bilayers, could be used for cell culture in order to utilise micro-impedance spectroscopy to examine further biological applications. To that purpose we have used normal epithelial cells from kidney (RPTEC) and a kidney cancer cell model (786-O). We demonstrate that the Tethapod system is compatible with the culture of 10,000 cells seeded to grow on a small area gold measurement electrode for several days without affecting the cell viability. Furthermore, the range of frequencies for EIS measurements were tuned to examine easily the characteristics of the cell monolayer. We demonstrate significant differences in the paracellular resistance pathway between normal and cancer kidney epithelial cells. Thus, we conclude that this device has advantages for the study of cultured cells that include (i) the configuration of measurement and reference electrodes across a microfluidic channel, and (ii) the small surface area of 6 parallel measurement electrodes (2.1 mm2) integrated in a microfluidic system. These characteristics might improve micro-impedance spectroscopy measurement techniques to provide a simple tool for further studies in the field of the patho-physiology of biological barriers.

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